MIF抑制剂ISO-1通过减轻氧化应激、炎症反应和细胞凋亡缓解脓毒症诱导的急性肾损伤  

MIF inhibitor ISO-1 alleviates sepsis-induced acute kidney injury by suppressing oxidative stress,inflammation,and apoptosis

作　　者：殷富康 张晓霞[1] 杨小军 李吉明[1] Yin Fukang;Zhang Xiaoxia;Yang Xiaojun;Li Jiming(Emergency Intensive Care Unit,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054)

机构地区：[1]新疆医科大学第一附属医院急救重症监护室(EICU),乌鲁木齐830054

出　　处：《安徽医科大学学报》2024年第12期2079-2086,共8页Acta Universitatis Medicinalis Anhui

基　　金：新疆维吾尔自治区自然科学基金资助项目(编号:2020D01C236)。

摘　　要：目的探究巨噬细胞移动抑制因子(MIF)抑制剂(S,R)-3-(4-羟苯基)-4,5-二氢-5-异噁唑乙酸甲酯(ISO-1)对脓毒症诱导的急性肾损伤(AKI)的作用及相关机制。方法将人肾小管上皮细胞系HK-2分为Con组(不做任何处理),ISO-1组(10μg/ml ISO-1处理24 h),LPS组(10μg/ml LPS处理24 h),LPS+ISO-1组(10μg/ml LPS处理24 h后加入10μg/ml ISO-1处理24 h)。ELSIA检测各组细胞上清液中肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)含量;6-羧基-2′,7′-二氯二氢荧光素二乙酸酯荧光指示剂(DCFH-DA)法检测细胞中活性氧(ROS)含量变化,TUNEL染色检测细胞凋亡,Western blot检测细胞中核转录因子E2相关因子2(Nrf2)、Kelch样环氧氯丙烷相关蛋白1(Keap1)、血红素加氧酶1(HO-1)和凋亡相关蛋白B淋巴细胞瘤-2基因产物(Bcl-2)、Bcl-2关联X蛋白(Bax)及裂解型半胱氨酸蛋白酶-3(c-caspase-3)的表达。采用盲肠结扎穿刺法(CLP)建立脓毒症小鼠模型,分为假手术(Sham)组,ISO-1对照(ISO-1)组,盲肠结扎穿刺(CLP)组和ISO-1治疗(CLP+ISO-1)组。实验结束后,取小鼠肾组织进行HE染色观察各组小鼠肾组织病理改变,检测血尿素氮(BUN)、血肌酐(Scr)和肾组织中髓过氧化酶(MPO)含量、谷胱甘肽(GSH)及超氧化物歧化酶(SOD)活性,并通过Western blot检测肾组织中上述MIF因子和Nrf2/Keap1信号通路和凋亡相关蛋白的表达。结果与Con组相比,LPS组和LPS+ISO-1组HK-2细胞中TNF-α、IL-1β、IL-6含量、TUNEL阳性率和细胞中ROS含量及Keap1、Bax与c-Caspase-3的蛋白表达均升高(P<0.05),而Nrf2、HO-1和Bcl-2蛋白降低(P<0.05);ISO-1组无明显变化(P>0.05)。与LPS组相比,LPS+ISO-1组细胞中TNF-α、IL-1β、IL-6含量、TUNEL阳性率和细胞中ROS含量及Keap1、Bax与c-Caspase-3的蛋白表达均降低(P<0.05),而Nrf2、HO-1和Bcl-2蛋白表达升高(P<0.05)。小鼠实验结果显示,与Sham组相比,CLP组与CLP+ISO-1组小鼠肾组织损伤严重,血清BUN、Scr含量及肾组织中Objective To investigate the effects and underlying mechanisms of the macrophage migration inhibitory factor(MIF)inhibitor(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazoleacetic acid methyl ester(ISO-1)on sepsis-induced acute kidney injury(AKI).Methods Human renal tubular epithelial HK-2 cells were divided into Con group(without any treatment),ISO-1 group(10μg/ml ISO-1 treatment for 24 h)and LPS group(10μg/ml LPS treatment for 24 h),LPS+ISO-1 group(10μg/ml LPS treatment for 24 h followed by 10μg/ml ISO-1 treatment for 24 h).ELISA was used to measure the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the cell supernatants.Reactive oxygen species(ROS)levels were assessed using the 6-carboxyl-2′,7′-dichlorodihydrofluorescein diacetate fluorescent indicator(DCFH-DA)method.Apoptosis levels were detected by TUNEL staining,and Western blot was employed to analyze the expression of proteins of Kelch like ECH associated protein 1(Keap1),NFE2 like bZIP transcription factor 2(Nrf2),heme oxygenase-1(HO-1),as well as apoptosis-related proteins Bcl-2,Bax,and cleaved Caspase-3(c-Caspase-3).A sepsis mouse model was established using the cecal ligation and puncture(CLP)method,and the mice were divided into four groups:sham-operated(Sham),ISO-1 control(ISO-1),CLP,and ISO-1 treatment(CLP+ISO-1).After the experiment,mouse kidney tissues were collected for HE staining to observe pathological changes.Blood urea nitrogen(BUN),serum creatinine(Scr),myeloperoxidase(MPO)levels in kidney tissues,glutathione(GSH)and superoxide dismutase(SOD)activities were measured.Western blot was also used to detect the expression of MIF and proteins in the Nrf2/Keap1 signaling pathway and apoptosis-related proteins in kidney tissues.Results Compared to the Con group,the LPS and LPS+ISO-1 groups showed significantly increased levels of TNF-α,IL-1β,IL-6,TUNEL-positive rates,ROS levels,and protein expressions of Keap1,Bax,and c-Caspase-3 in HK-2 cells(P<0.05),while the expressions of Nrf2,HO-1,and Bcl-

关 键 词：脓毒症 急性肾损伤 巨噬细胞移动抑制因子拮抗剂ISO-1 氧化应激 炎症 细胞凋亡 

分 类 号：R73[医药卫生—肿瘤] R914[医药卫生—临床医学]